Morphological and transcriptomic responses/acclimations of erect‐type submerged macrophyte Hydrilla verticillata both at low‐light exposure and light recovery phases

Abstract Light intensity is a determinant for submerged macrophytes. Little is known about their molecular responses to low‐light exposure, despite more informative and responsive than morphological traits. For erect‐type submerged macrophytes, the stem is more crucial relative to the leaf in acclimation to low‐light stress, but receives less attention. We determined morphological and stem transcriptomic responses/acclimations of Hydrilla verticillata to extremely and mildly low light (7.2 and 36 μmol photons m−2 s−1, respectively), that is, EL and ML, with the radiation intensity of 180 μmol photons m−2 s−1 as the control. Low‐light exposure continued for 9 days, followed by a 7‐day recovery phase (180 μmol photons m−2 s−1). At the exposure phase, the low‐light treatments, in particular the EL, decreased the relative growth ratio, but induced greater height and longer stem internode distance and epidermal cell. Such responses/acclimations continued into the recovery phase, despite more or less changes in the magnitude. Transcriptome showed that the photosynthetic system was inhibited at the exposure phase, but the macrophyte adjusted hormone synthesis relating to cell division and elongation. Moreover, the EL activated cell stress responses such as DNA repair. Following light recovery, the macrophyte exhibited a strong‐light response, although energy metabolism enhanced. Especially, the EL enriched the pathways relating to anthocyanin synthesis at such phase, indicating an activation of photoprotective mechanism. Our findings suggest that negative influences of low light occur at both low‐light exposure and recovery phases, but submerged macrophytes would acclimate to light environments. Transcriptome can show molecular basis of plant responses/acclimations, including but not limited to morphology. This study establishes a bridge connecting morphological and molecular responses/acclimations.


| INTRODUC TI ON
Submerged macrophytes play an irreplaceable role in maintaining the structure and functions of shallow lake ecosystems (Scheffer et al., 2001), which provide food for heterotrophic organisms, and also serve as shelter for a large number of animals (Paice et al., 2017;Wang et al., 2023).Moreover, the macrophytes can absorb excessive nutrients, inhibit algal growth, stabilize the sediment, and release oxygen (Liu et al., 2020;Paice et al., 2017), and thus help maintain shallow lake in the clear water state (Scheffer et al., 2001).For aquatic ecosystem health, therefore, the growth performance of submerged macrophytes is considered as a main indicator.Light intensity, a critical determinant for plant growth and metabolism, plays a decisive role in controlling the productivity and distribution of submerged macrophytes (Rao et al., 2021).
Water level rise, as well as turbidity increase, generally induces low-light exposure on submerged macrophytes, and such scenario is becoming more frequent due to extreme precipitation events, eutrophication, and reservoir construction (Munoz et al., 2018;Voesenek & Bailey-Serres, 2015).Numerous studies have shown that low-light environments generally inhibit the growth of submerged macrophytes (Gao et al., 2021;Yuan et al., 2016), though they have the ability of acclimatized adjustment (Wang et al., 2021;Yang et al., 2022).After elimination of the low-light exposure, these macrophytes may gradually return to a normal state, or alternatively, continue to maintain a low growth rate, due to the legacy of low-light exposure (Ibanez et al., 2012).For community stability and ecosystem resistance, whether and how the submerged macrophytes recover from the low-light damage are remarkable issues.
Morphological traits, such as plant height, leaf number, root:shoot ratios, and cell length, are measurable properties relating to productivity and acclimation to the environment (He et al., 2019).
They contain a great deal of information involving many patterns and processes, such as phylogenetic signals, physiological functions, and environmental constraints, at a wide range of scales (Visakorpi et al., 2023).Thus, the morphological plasticity is widely adopted to investigate the vulnerability of macrophytes to environmental change.For instance, submerged macrophytes are found to adjust morphological traits to light shortage, such as decreasing dry matter contents, thinning and elongating stems, and increasing leaf number (Chen et al., 2020).However, the morphological responses/acclimations often delay, and even are undetectable and variable (Chen et al., 2020;van der Plas et al., 2020).By contrast, the gene expression generally responds quickly to external interference and acts as the molecular basis of morphological plasticity (Akman et al., 2016;Whitehead, 2012).Transcriptome analyses can provide insights into gene expression information, and help build a bridge between gene expression and morphological performance.By analyzing the whole transcriptome of Posidonia oceanica, Ruocco et al. (2021) found several epigenetic mechanisms, and suggested that the cross-talk between DNA methylation and the cellular energetic status regulates shoot metabolism at low irradiance levels.Davey et al. (2018) demonstrate that Zostera muelleri tends to amplify the expression of genes, relating to photosynthesis, carbohydrate metabolism, and abscisic synthesis, to cope with low-light exposure.Thus, transcriptome analysis is an effective measure for revealing plant acclimation strategy under unfavorable conditions.
In contrast with the leaf, the stem receives limited attention when exploring how submerged macrophytes acclimate to environmental changes.The truth is, though, the stem also plays a pivotal role in plant acclimation to low-light environments (Sun et al., 2004).
Especially for erect-type submerged macrophytes, the stem helps the leaf capture light energy by elongating toward the water surface, but also participates directly in primary production (Liu et al., 2021).
Therefore, the responses of stem should be taken into account to unravel macrophyte acclimation to low-light exposure.Hydrilla verticillata, a typical erect-type submerged macrophyte, is widely distributed in freshwater ecosystems in Eurasia.However, relatively little is known about how the stem acclimates to declining light intensity.
Here, based on a microcosm experiment, we determined stem transcriptomic responses/acclimations, as well as morphological responses/acclimations, to low-light exposure, and also concerned the performance after recovery of light conditions.For this study, we hypothesized that (1) H. verticillate would cope with low-light exposure by morphological acclimations; (2) the growth of H. verticillata cannot well return to normal after the recovery of light intensity; and (3) transcriptome can provide detailed information on responses/acclimations, including but not limited to the molecular basis of morphological performances.

| Experimental design
Hydrilla verticillata shoots were collected from Lake Taihu (31°20′47″ N, 120°28′41″ E), a shallow lake with a water area of 2338 km 2 and an average water depth of 1.9 m.The top 8-cm segments were cut from healthy individuals, weighed, and used as the asexual propagules in this experiment.After rinsed with distilled water, the propagules were planted in plastic bottles (10 cm diameter, 40 cm height, and eight propagules in each bottle) containing 2 L distilled water and 200 g sediment, with total organic carbon 12.8 mg g −1 , total nitrogen 2.73 mg g −1 , and total phosphorus 0.730 mg g −1 .These propagules in bottles were pre-incubated at 27°C for 15 days in three growth chambers, with the light-dark regime of 12 h:12 h and the radiation intensity of 180 μmol photons m −2 s −1 .Following the pre-incubation, based on the light compensation point of H. verticillata (i.e., 15.8 μmol photons m −2 s −1 , Su et al., 2004), the light intensity of two chambers were, respectively, lowered to 7.2 and 36 μmol photons m −2 s −1 .These two chambers represented an extremely low-light environment (EL) and a mildly low-light environment (ML), respectively, with another chamber still maintained the light intensity at 180 μmol photons m −2 s −1 (CK).
According to the mean duration of flood in Lake Taihu in the past 30 years, the low-light exposure continued for 9 days in this study.
Subsequently, the light intensity of both EL and ML treatments returned to 180 μmol photons m −2 s −1 , and the recovery phase continued for 7 days.For this experiment, there were six bottles evenly placed in each growth chamber, and transposition was carried out every day to avoid unequal light radiation.Moreover, distilled water was supplemented as needed to maintain a constant water level throughout the experiment.

| Determination of morphological traits
At the end of both low-light exposure and light recovery phases, we chose three replicated plastic bottles from each growth chamber and determined morphological parameters of H. verticillata.Two healthy individuals from each plastic bottle were harvested, washed, and dried with filter paper to determine the total biomass, the number of stem internode, and the average distance of stem internode.
In addition, plant height and stem diameter (at 5 cm down the tip) were measured with a ruler (0.1 cm precision) and a micrometer (0.02 mm precision), respectively.After the measurement, the H. verticillata individuals in each plastic bottle were separated into leaves, stems, and roots, oven-dried at 80°C to a constant weight, and then weighed to obtain the dry weight.The stem mass ratio was defined as the ratio of stem dry weight to total dry weight.Relative growth rate (RGR) was calculated as where W 1 is the initial fresh weight (mg), W 2 is the final fresh weight (mg), and ∆T is the time (day).In addition, a scan with the electron microscope (JSM-5610LV, Thermo Fisher Scientific) was used to determine stem epidermal cell length (at least 15 cells per sample) at 5 cm down the tip.

| RNA extraction and sequencing
In addition to the morphological parameters, the apical 5-cm stem of H. verticillata was clipped, shock-frozen in liquid nitrogen, stored at −80°C, and used for transcriptome analysis.Total RNA was extracted from the stem samples with RNA 6000 Nano kit (Agilent).
RNA concentration and purity were determined by UV spectrophotometry, and the integrity was assessed by an Agilent 2100 Bioanalyzer (Agilent).The total amount of RNA required for a single library construction is ≥1 μg, the concentration is ≥35 ng/μL, OD 260 / 280 is ≥1.8, and OD 260 / 230 is ≥1.0.In this experiment, all RIN (RNA Integrity) numbers were above eight, indicating that high-quality material was used for sequencing.Fifteen indexed cDNA libraries, that is, (three light treatments at the end of the low-light exposure phase + two low-light treatments at the end of light recovery phase) × three replicates, were constructed and sequenced with an Illumina Novaseq 6000 platform (Paired-ends 2 × 150 cycles).

| Transcriptome assemble and functional annotation
Samples were collected at the end of both low-light exposure and light recovery phases, and transcripts were spliced after each sampling.For this study, the cDNA libraries of CK treatment constructed at the end of the low-light exposure phase were as the control both at the low-light exposure and recovery phases.Sequence raw reads were checked for data quality using FastQC.Cutadapt was used to remove sequences with adapters at the 3′ end and reads with an average quality score below Q20.We used Trinity with default parameters to assemble clean reads into a transcriptome, as there is no available reference genome for H. verticillata.The longest transcript was selected and used for subsequent analysis.Benchmarking Universal Single-copy Orthologs (BUSCOs) was used to complete the integrity of the assembled transcriptome.

| Gene expression and functional enrichment analyses
RSEM with default parameters was used to estimate the gene expression level by calculating the read count of each transcript.We used "edge R" package to perform differential analysis of highabundance genes (≥1 read/count per million).Transcripts were considered differentially expressed when FDR < 0.05 and logFC > ±1.

| Statistical analysis
All statistical analyses were performed with R (v. 4.2.3,R Core Team, 2023) with an accepted significance level of p < .05.The normality of all data was tested with the Shapiro-Wilk test before statistical analysis.Repeated measures analysis of variance (ANOVA) was used to assess the effects of light treatment, incubation phase, and their interactions on morphological traits.Following one-way ANOVA, Tukey's multiple comparison test was used to detect the differences in plant height, the number of stem internode, the average distance of stem internode, stem diameter, stem mass ratio, fresh weight, and RGR among light treatments at the same phase, that is, the low-light exposure phase or the light recovery phase.

| Morphological traits
Light irradiance intensity generally exerted significant effects on morphological traits, and the effects on plant height and stem internode number did not vary significantly between the low-light exposure and the recovery phases (Table 1; Figure 1).At the end of low-light exposure phase, the decreased light irradiance, especially the EL treatment, induced significant increases in plant height, stem internode distance, stem mass ratio, as well as significant decreases in internode number, fresh weight, stem diameter, and RGR (Figure 1).Moreover, the length of stem epidermal cells obviously increased with the decrease of light intensity (Figure 2a-c).It is worth noting that such influences from low light continued into the light recovery phase, although the magnitudes had more or less changes (Figures 1 and 2d,e).

| Transcript assembly and annotation results
In this project, a total of 65 million clean reads were produced, with 3.7~4.8 million per sample (Table A1 in Appendix).At the end of the low-light exposure phase, there are 101,363 unigenes obtained, with a mean length of 876.71 bp and an N50 value of 1268 bp (Table A2 in Appendix).The functional annotation analysis showed that 41,027 (40.48%) and 18,303 (18.06%) unigenes significantly matched NR and KEGG databases, respectively (Table A3 in Appendix).There was a high proportion (86.5%) of complete BUSCOs present, indicating the transcriptome was relatively complete (Figure A1 in Appendix).
At the end of light recovery phase, 117,484 unigenes were observed, with a mean length of 826.74 bp and an N50 value of 1121 bp (Table A2 in Appendix).There were 49,745 (42.34%) and 25,434 (21.65%) unigenes significantly matching NR and KEGG databases, respectively (Table A3 in Appendix).Moreover, the proportion of complete BUSCOs was up to 84.3% (Figure A1 in Appendix).

| DEGs analysis
In contrast with the ML treatment, the EL treatment induced a larger number of DEGs both at the low-light exposure and light recovery phases (Figure 3).
Nine days into the low-light exposure phase, a total of 814 DEGs comprising 456 (56.0%) and 338 (44.0%) down-and up-regulated genes, respectively, were detected in ML versus CK (Figure 3c).
Seven days into the light recovery phase, there were 164 (50.0%) and 310 (57.0%) up-regulated genes in ML and EL treatments, respectively, while the number of down-regulated genes were, respectively, 164 (50.0%) and 244 (43.0%) (Figure 3d).In the DEGs, there were 137 (28.9%) up-regulated and 111 (27.2%) down-regulated genes shared by two low-light treatments (Figure 3d).The PCoA showed that the first axis (44.54% of total variance) mainly separated low-light treatments from CK, while the difference in the second axis (22.23% of total variance) was caused by the intra-group differences in the samples (Figure 3b).

TA B L E 1
Results (F-values) of repeated measures ANOVA indicating effects of incubation phase, light treatment, and their interaction on morphological traits.

| KEGG pathway analysis of DEGs
At the end of low-light exposure phase, 814 DEGs were significantly enriched (FDR < 0.05) into eight pathways in the ML treatment, most of which relate to metabolism, such as photosynthesis, photosynthesis-antenna proteins, and carbon fixation in photosynthetic organisms.In addition, plant hormone signaling transduction (FDR = 0.094) and flavonoid biosynthesis (FDR = 0.18) were also enriched (Figure 4a; Table A4 in Appendix).A similar pattern was also found in the EL treatment, with six pathways significantly affected, including the pathways regulating energy metabolism, carbohydrate metabolism environmental acclimation (circadian rhythm-plant), and biosynthesis of other secondary metabolites.The DNA repair was enriched (FDR = 0.068) in the EL treatment (Figure 4b; Table A4 in Appendix).In terms of the genes involving in photosynthesis, the genes encoding PsbO and PsbQ proteins were down-regulated in both low-light treatments (Figure 5a).However, the genes encoding LHCII were down-regulated and up-regulated, respectively, in the ML and EL treatments (Figure 5a).Table A5 in Appendix).In addition, we found that genes relating to anthocyanin synthesis were significantly down-regulated in the EL treatment, including BZ1, ANS, and F3H (Figure 6a).

F I G U R E 5
The expression of genes encoding some proteins at the low-light exposure phase.
Underwater light intensity is a critical determinant for the survival and growth of submerged macrophytes.Based on an incubation experiment, we investigated morphological and molecular responses/acclimations of H. verticillata to low-light exposure and also paid attention to the performance after elimination of lowlight exposure.We found the morphological traits and the transcriptome expression generally varied with light treatments at both the exposure and the recovery phases (Figure 7).Moreover, the transcriptomic information not only provided the molecular basis of plant morphological traits, but also included unobservable response mechanisms (Figure 7).To the best of our knowledge, this is the first attempt to explore the molecular mechanism behind how submerged macrophytes acclimate the stem for lowlight environments.

| Morphological responses/acclimations to low-light environments
In this study, the low-light exposure, especially when the light intensity was below the light compensation point, substantially inhibited the growth of H. verticillata, just as shown by the low biomass and RGR.This is consistent with the results of many previous

F I G U R E 6
The expression of genes encoding some proteins at the light recovery phase.
When the photosynthetic function of stem is taken into account, such morphological acclimation is becoming more important for the erect-type submerged macrophytes.
After 7 days of light recovery, the growth of H. verticillata did not well return to normal, which is consistent with our second hypothesis.In general, the morphological difference between low-light treatments and the control obviously remained.For example, there was no significant change in terms of RGR in the EL treatment, which was even lower than that at the low-light exposure phase in the ML  of photosystem II and thus a decrease in photosynthetic capacity (Wei et al., 2016).Moreover, the expression of the gene encoding ppc protein was significantly down-regulated in low-light treatments.
As ppc enzymes play an important role in nocturnal CO 2 assimilation and daytime CO 2 absorption cycles (Boxall et al., 2020), the present results mean that low light interferes with carbon fixation and energy absorption.Therefore, these findings provided the molecular basis for the inhibition effect of low light on plant growth performance.Phytohormones control plant growth by regulating gene expression, which is particularly important when plants cope with unfavorable light environments (Curaba et al., 2014).In this study, the low light affected the synthesis of several proteins, such as EBF1/2, ABF, and GH3, in the plant hormone signaling pathway.As EBF1/2 protein is a key component of the ethylene synthesis pathway, the synthesis reduction of which is beneficial to ethylene accumulation in plants, thus promoting elongation of stem internode for submerged macrophytes (Kendrick & Chang, 2008;Kuroh et al., 2018).Abscisic acid is an important plant hormone relating to various aspects of plant development, such as inhibition of cell division and growth.In the synthesis of abscisic acid, the ABF protein acts as a key transcription regulation factor.Therefore, the down-regulated expression of the transcript encoding the ABF protein is beneficial to the improvement of H. verticillata stem growth under low-light conditions.It is also noteworthy that the transcripts encoding a protein associated with auxin synthesis (GH3) were overexpressed in the EL treatment, which implies oversynthesis of auxin.The auxin is considered to promote the elongation of plant cells, but also plays a regulatory role in the distribution of carbohydrates and energy (Alpert et al., 2002;Javid et al., 2011).Under low-light conditions, the increase of auxin synthesis can help the plant concentrate energy and carbohydrates into the tip meristem, thus promoting stem elongation.These findings above indicate hormone adjustment in response to low-light exposure, and further provide a molecular basis for growth performance.
In addition to phytohormones, the secondary metabolites also play a critical role for plants in coping with environmental stress (Erb & Kliebenstein, 2020;Jan et al., 2021).As a common secondary metabolite, flavonoids have multiple functions in physiological metabolism, such as resisting environmental stress and self-protection (Harborne & Williams, 2000;Nakabayashi & Saito, 2015).In the flavonoid biosynthesis pathway, CHS, DFR, ANS, and ANR are the major intermediate enzymes (Tan et al., 2013).The expression levels of synthetic transcripts encoding these enzymes were down-regulated under both low-light treatments, implying that the low-light exposure reduces the synthesis of flavonoids, and thus induces a decline in defense against environmental disturbance.Consistent with our study, previous studies regarding terrestrial plants also found shade treatment reduced the synthesis of flavonoids (Ye et al., 2021).In addition, the pathways relating to environmental acclimation, such as circadian rhythm-plant, were uniquely inhibited in the EL treatment.The response of flavonoid synthesis suggests that the environmental adaptability of H. verticillata would greatly decrease when the light intensity is lower than the light compensation point.These findings suggest that low-light exposure would decrease the defense capability of submerged macrophytes, and therefore the plant population would suffer worse impacts when other environmental disturbances happen simultaneously with the low-light exposure.
In the EL treatment, we enriched certain pathways relating to cellular stress response, such as DNA repair.In the DNA repair, all transcripts associated with the small chromosome maintenance complex (MCM2-7) were significantly up-regulated in the EL treatment.Considering that MCM takes part in DNA replication by controlling cell mitosis at the S-phase (Chang et al., 2007;Shultz et al., 2009), such results indicate a strengthening of DNA respiration under extremely low-light conditions.These results indicate that the submerged macrophytes would cope with low-light environments by active cellular stress response, which cannot be found in studies only involving morphological traits.Collectively, these results above support our third hypothesis that transcriptome can provide detailed information on responses/acclimations, including but not limited to the molecular basis of morphological performances.

| Transcriptomic responses/acclimations at the light recovery phase
Oxidative phosphorylation is a coupling reaction, in which the energy released during the oxidation of substances supplies ADP and inorganic phosphoric acid to synthesize ATP (Cogliati et al., 2016;Fuchs et al., 2020).Specifically, the expression of the gene encoding inorganic pyrophosphatase was up-regulated in ML treatment, indicating an over-synthesis of ATP (Chu et al., 2020).In addition, the pathways associated with carbohydrate metabolism were significantly enriched in ML treatment, such as glycolysis/gluconeogenesis and fructose and mannose metabolism.As one main metabolic pathway of glucose, glycolysis can provide ATP for life activities (Nie et al., 2020).Given that glucose-6-phosphate isomerase (GPI) controls the second step of glycolysis, that is, catalyzing the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate (Haller et al., 2011;Lin et al., 2009), the up-regulated expression of the gene encoding GPI implies an enhanced glycolysis.Therefore, these results suggest that the recovery of light intensity would boost energy metabolism, and thus contributing to plant growth and recovery.
Despite a potential promotion on plant growth, the shift from low light to normal light exerted substantial disruptions on the photosynthetic system.Non-photochemical quenching is an important self-protective mechanism for plants exposed to strong light, where PsbS protein is considered to be the key (Ruban, 2016).The gene encoding PsbS protein was significantly up-regulated in both lowlight treatments, indicating that the shift of light intensity causes damage to the photosynthetic system of H. verticillata stem.As a core component of PSII, moreover, the reduction of pabW protein synthesis indicates reduced stability of PSII and inhibited photosynthesis (Bishop et al., 2003;Shi et al., 2000).Similarly, Hussner et al. (2010) also found the photosynthetic system of submerged macrophytes growing in a low-light environment was inhibited when they were transferred to a strong-light environment.Moreover, the transcriptomic information also indicates a self-protection of H. verticillata when it suffers sharp changes in light environments.
In summary, the submerged macrophytes would be subjected to light damage when the light environment recoveries from a lowlight state, and make acclimatized adjustments to such environment transformation, which may be the reason behind the poor morphological performance of H. verticillata at the light recovery phase.
Such founding confirms our second hypothesis at the molecular level, that is, H. verticillata cannot well return to normal after the recovery of light intensity.
In addition, we also found several unique pathways in the EL treatment.The pathways relating to anthocyanin synthesis, such as flavonoid biosynthesis and anthocyanin biosynthesis, were activated, and the expression of three key protein-coding genes, including BZ1, ANS, and F3H, were significantly up-regulated.Considering that anthocyanins are key pigments protecting plant cells against photodamage (Huang et al., 2019), the pathways and genes mentioned above indicate an activation of photo-protective mechanisms of submerged macrophytes when the light intensity returns to normal level.In addition, the MAPK signaling pathway plant was also significantly enriched.In a previous study (Mizoguchi et al., 1997), the MAPK cascades have been found to take part in higher plants acclimation to environmental stresses.These unique pathways indicate that the submerged macrophytes subjected to extremely lowlight exposure would invest in the photo-protection when the light conditions returned to presumably which is a significant cause of why these plants had limited increase in the fresh weight at the light recovery phase.

| CON CLUS IONS
In  : Arrange all the sequences from the longest to the shortest, add the sequence lengths in that order, and when the sum reaches 50% of the total length of the sequence, the length of the last sequence; N50 (bp): Arrange all the sequences in order of length from longest to shortest, add the sequence lengths in that order, and when the length of the sum reaches 90% of the total length of the sequence, the length of the last sequence; N50 Sequence No.: The total number of sequences with length greater than N50; N90 Sequence No.: The total number of sequences with length greater than N90; GC%: GC content of the sequence.

TA B L E A 2
Trinity assemble result analysis.
TA B L E A 3 Annotation results summary.
The differentially expressed genes (DEGs) were further analyzed by KEGG enrichment analysis with the online platform (https:// www.omics hare.com/tools).The ggord package based on R was used for Principal Co-ordinates Aanalysis (PCoA) to reflect the similarity between samples.Venn diagram (http://www.ehbio.com/test/venn/#/) was used to show the distribution of the number of DEGs that were unique and shared by ML versus CK and EL versus CK at the low-light exposure and light recovery phases.The heatmap showed the expression levels of genes in KEGG pathways at the low-light exposure and light recovery phases.Transcriptomics data used in this study are publicly available from NCBI (BioProject ID PRJNA977960).

F
I G U R E 1 Responses of plant height (a), internodes number (b), stem internodes distance (c), stem diameter (d), fresh weight (e), RGR (f), stem mass ratio (g) to light intensity at the low-light exposure and light recovery phases.Data are means and error bars are SD (n = 3).Different lowercase letters indicate significant differences between light irradiance treatments at the same phase (p < .05).F I G U R E 2 SEM micrograph showing the stem epidermal cell length of Hydrilla verticillate at the low-light exposure (a: CK; b: ML; c: EL) and light recovery phases (d: CK; e: ML; f: EL).F I G U R E 3 Analysis of gene expression in experimental Hydrilla verticillatac plants.PCoA was performed with the normalized expression values of plant genes at the low-light exposure (a) and light recovery phases (b).Venn diagram depicting shared and unique DEGs in different light treatments at the low-light exposure (c) and light recovery (d) phases.Bold values denote the DEG number, while gray values above and below the bold value indicate up-regulated and down-regulated DEGs.
Figure 4b; TableA4in Appendix).In addition, all genes identified involving in DNA repair were up-regulated in the EL treatment, including six DNA replication licensing factor MCM (2-7) genes (Figure5b;Table A4 in Appendix).At the end of the light recovery phase, 328 DEGs were significantly enriched into seven KEGG pathways in the ML treatment, including photosynthesis-antenna proteins, flavonoid biosynthesis, circadian rhythm-plant, anthocyanin biosynthesis, and MAPK signaling pathway-plant (Figure4c; TableA5in Appendix).In the EL treatment, 554 genes were enriched into 9 KEGG pathways.The Glycolysis/Gluconeogenesis was marginally significantly enriched (FDR = 0.060; Figure4d; TableA5in Appendix).Twenty-two and 16 genes involving in photosynthesis were identified as differentially studies regarding the effects of light intensity on submerged macrophytes(Chen et al., 2020;Yuan et al., 2016).For example,Yang et al. (2022) found that the biomass of Vallisneria natans decreased with the decline of light intensity, and more resources were devoted to clonal reproduction and population stability than to primary production.The decreased photosynthetic products under low-light conditions can take account for such a phenomenon(He   et al., 2019).In addition, plants often synthesize more secondary substances to acclimate to harsh environments, thus further decreasing the matter and energy allocations to growth.Nevertheless, we also found H. verticillata positively coped with the low-light environment in this study.Specifically, H. verticillata in low-light treatments had higher shoots, longer internode distance, thinner stem, longer stem epidemic cell length, and greater stem mass ratio, which could help capture sunlight, thus alleviating lowlight stress(Fu et al., 2012;Strand & Weisner, 2001).Such findings are consistent with our first hypothesis, in which H. verticillata is expected to cope with low-light exposure by morphological acclimations.Similar results have been found in previous studies on canopy-type submerged macrophytes (e.g.,Chen et al., 2020;Fu et al., 2012).For example,Wang et al. (2021) found Potamogeton crispus had high plant height, large leaf area, and long leaf length, as well as thin stem in low-light environments.With the so-called "light foraging" strategy, the submerged macrophytes can get amounts of light energy at minimal cost under low-light conditions.
treatment.Presumably, the reason behind such insensitivity may lie in that the plants have no enough energy to acclimate their morphology in time to light recovery, due to extremely low energy storage at the low-light exposure phase.Collectively, our findings indicate that low-light exposure inhibits the growth of erect-type submerged macrophytes, especially when the light intensity is lower than the light compensation point, in spite of morphological acclimation.Moreover, such inhibition effects can continue into the light recovery phase.

4. 2 |
Transcriptomic responses/acclimations to light irradiance 4.2.1 | Transcriptomic responses/acclimations at the low right exposure phase In this study, low-light exposure significantly affected the photosynthetic capacity of H. verticillata.Specifically, as essential proteins for the optimal oxidation activity of photosystem II, the reduced synthesis of PsbO and PsbQ in low-light treatments indicates an inhibition

F
Diagram showing the multi-level responses/acclimations of Hydrilla verticillata to light treatments.
this study, we determined morphological and stem molecular response/acclimation of H. verticillata to low-light exposure both at the low-light exposure and light recovery phases.At the exposure phase, the low-light treatments, especially when the light intensity is lower than the light compensation point, significantly decreased plant fresh weight and RGR, but increased plant height, stem internode distance, stem mass ratio, and stem epidermal cell length.The transcriptome information showed that the low light induced a reduction of photosynthetic capacity, which is in accord with the low fresh weight and RGR, while the up-regulated pathways relating to the synthesis of phytohormones provided molecular basis for the increased plant height and internode distance.Moreover, the pathways relating to cell stress response such as DNA repair were enriched in the extremely low-light treatment.At the light recovery phase, the plants from the low-light treatments still had a low RGR, though the response of transcriptome showed an enhancement of energy metabolism.Moreover, the transcriptome information implies that the shift from low light to normal light exerted damage on the photosynthetic system and induced plant self-protection.In addition, the enriched pathways relating to anthocyanin synthesis, following the shift from extremely low light to normal light, indicate an activation of photo-protective mechanism.Our findings indicate the negative influences of low light on submerged macrophytes not only occur at the low-light exposure phases but also after light recovery.Nevertheless, the macrophytes would adjust morphological traits to surrounding light environments.The transcriptome can show the detailed molecular basis of plant responses/acclimations, including but not limited to morphological performances.This study establishes a bridge connecting morphological and molecular responses/ acclimations and contributes to aquatic ecosystem management and Data filtering statistics.
Database: type of database; Number: represents the number of successfully annotated unigenes in the database; Percentage (%): Represents the ratio of the number of unigenes successfully annotated in the database to the total number of unigenes; In all database: The number of unigenes annotated in all databases.Top 20 list of enriched KEGG pathway in ML and EL treatment in low-light exposure phase.